Project Summary Recent advances with induced pluripotent stem (iPS) cells and tissue engineering have opened the door to keratinocyte-based tissue replacement for patients with Recessive Dystrophic Epidermolysis bullosa (RDEB). While our lab has shown that bone morphogenic protein (BMP) and retinoic acid (RA) morphogens can induce ES/iPS-derived graftable human keratinocytes, detailed mechanistic insights into keratinocyte differentiation remain a major roadblock to efficient tissue manufacturing and a goal of the NIH Regenerative Medicine Innovation Project. Without RA/BMP, master regulator p63 binds but exhibits few transcriptional changes, highlighting the importance of morphogen-lineage selector interactions. HiChIP analysis shows that RA/BMP induces chromatin conformational changes that connect chromatin-bound p63 to linked enhancers/promoters and determine transcriptional specificity at each gene. Through our novel network transcription factor (TF) inference model we discovered that forced expression of a single transcription factor, TFAP2C, can induce functional keratinocytes in the absence of RA/BMP. We subsequently identified a two-step mechanism where the TFAP2C initiates the simple epithelial landscape, and induces expression of and opens additional binding sites for the p63. In turn, p63 matures the TFAP2C-patterned epigenetic landscape resulting in p63 positive autoregulation and the closing of a subset of TFAP2C binding sites, shifting the landscape to a p63-centric keratinocyte TF network. These data support the intriguing hypothesis that RA/BMP initiates epigenetic changes through TFAP2 family members that ultimately result in p63-driven epigenetic maturation of the landscape to keratinocytes. In this proposal we aim to address key gaps in our knowledge through: Functional validation of the TFAP2- centric network in epigenetic landscape initiation by confirming TFAP2 necessity for RA/BMP- mediated epigenetic landscape change, determining TFAP2C functional requirement for the early TF network, and defining the morphogen-inducible TFAP2C interactome; Elucidation how p63 matures the chromatin landscape during keratinocyte production by determining how TFAP2 and p63 cooperate to allow p63 positive autoregulation, determining if keratinocyte maturation requires p63- dependent TFAP2 repression, and completing and validating an inference TF model of keratinocyte differentiation. Successful completion of this proposal will provide deep mechanistic insights into the chromatin dynamics of tissue differentiation and establish a detailed epigenetic characterization that enables development of our novel cell therapy for a previously untreatable genetic skin disorder.